FY2023 Annual Report

Nucleic Acid Chemistry and Engineering Unit
Professor Yohei Yokobayashi

 

Abstract

In FY2023, we published the results of a long-term collaboration with Astellas Pharma on the development of novel small molecule aptamers for mammalian riboswitch applications. A patent application was filed on the technology based on the discovery. Another work on small RNA ligase ribozymes that are relevant to the origin of life research was published. Meanwhile, we have made progress in further development of mammalian riboswitches, cell-free riboswitch applications, and structural and chemical biological analysis of new aptamers which will be published in the near future.

1. Staff

 

  • Dr. Narae Kim, Researcher
  • Dr. Harikrishnan K. S., Researcher
  • Dr. Thao Le, Researcher
  • Dr. Yoko Nomura, Science and Technology Associate
  • Dr. Lara Sellés Vidal, Researcher
  • Dr. Bochen Zhu, Researcher
  • Tomoya Noma, Graduate Student
  • Nao Miyahira, Technical Staff
  • Yayoi Maehara, Technical Staff
  • Dr. Haifeng Zhang, Technical Staff
  • Kyoko Chinen, Research Unit Administrator

(As of 3/31/2023)

2. Collaborations

2.1 Collaboration with an industrial partner

  • Undisclosed.

2.2 AMED-BINDS Program

  • Undisclosed collaboration on aptamer characterization and applications.
  • Type of collaboration: Joint research

3. Activities and Findings

3.1 Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals

Synthetic riboswitches that can regulate gene expression by a small molecule recognized by an RNA aptamer in mammalian cells have various potential applications in biotechnology and medicine. However, the variety of small molecules and their cognate aptamers that have been demonstrated to function in mammalian cells is limited. The currently available aptamer-ligand pairs also require high small molecule concentrations to enable gene regulation, making them less desirable for industrial and biomedical applications. We conducted in vitro selection of RNA aptamers against a small molecule ASP7967 whose structure is closely related to ASP2905, a known inhibitor of potassium voltage-gated channel sub-family H member 3 (KCNH3). One of the aptamers selected (AC17–4) was found to be functional in HEK293 cells, and it was used to design aptazyme-based riboswitches that can activate gene expression (>10-fold) in the presence of ASP2905 or ASP7967 at as low as 5 μM in the culture medium. An aptazyme-based riboswitch was successfully used to regulate human erythropoietin expression in mice injected with an adeno-associated virus (AAV8) vector using orally administered ASP7967. Furthermore, by combining aptazyme-based and exon-skipping riboswitch mechanisms, an ON/OFF ratio approaching 300 was achieved with a low basal expression level in cultured cells.

Figure 1: Schematic illustration of the new aptamer and riboswitch mechanism.

 

 

3.2 RNA ligase ribozymes with a small catalytic core

Catalytic RNAs, or ribozymes, catalyze diverse chemical reactions that could have sustained primordial life in the hypothetical RNA world. Many natural ribozymes and laboratory evolved ribozymes exhibit efficient catalysis mediated by elaborate catalytic cores within complex tertiary structures. However, such complex RNA structures and sequences are unlikely to have emerged by chance during the earliest phase of chemical evolution. Here, we explored simple and small ribozyme motifs capable of ligating two RNA fragments in a template-directed fashion (ligase ribozymes). One-round selection of small ligase ribozymes followed by deep sequencing revealed a ligase ribozyme motif comprising a three-nucleotide loop opposite to the ligation junction. The observed ligation was magnesium(II) dependent and appears to form a 2′–5′ phosphodiester linkage. The fact that such a small RNA motif can function as a catalyst supports a scenario in which RNA or other primordial nucleic acids played a central role in chemical evolution of life.

Figure 2: Small RNA ligase (N8-1) ribozyme structure and activity.

 

4. Publications

4.1 Journals

  1. Nomura Y, Yokobayashi Y, RNA ligase ribozymes with a small catalytic core. Sci Rep 2023; 13: 8584. (PubMed / Journal (Open Access))
  2. Fukunaga K, Dhamodharan V, Miyahira N, Nomura Y, Mustafina K, Oosumi Y, Takayama K, Kanai A, Yokobayashi Y, Small-Molecule Aptamer for Regulating RNA functions in Mammalian Cells and Animals. J Am Chem Soc 2023; 145: 7820-7828. (PubMed / Journal (Open Access)) (OIST press release)

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

  1. Yokobayash, Y., Fukunaga, K., Dhamodharan, V., Miyahira, N., Nomura, Y., Oosumi, Y., Takayama, K., Kanai, A. A Novel Small Molecule-Aptamer Pair for Regulating Gene Expression In Vitro and In Vivo, ASGCT 26th Annual Meeting, (Los Angeles, U.S.A.), May 17 (2023). (Oral)
  2. Sellés Vidal, L., Suzuki, M., Yokobayashi, Y. Conditional transgene expression triggered by specific mRNA sequence, 2023 Synthetic Biology: Engineering, Evolution & Design (SEED), (Los Angeles, U.S.A.), May 30 (2023). (Poster)
  3. Noma, T., Yokobayashi, Y. Development of Cell-type Specific Mammalian Gene Switches via Directed Evolution of group I self-splicing ribozyme, ERATO Evolving Symbiosis Project Okinawa, (Okinawa, Japan), May 24 (2023). (Poster)
  4. Yokobayashi, Y. 「セルフリーリボスイッチの開発とその応用」, オンチップ・バイオテクノロジーズ社オリジナルウェビナー (Online), Jul 20 (2023). (Oral)
  5. Noma, T., Yokobayashi, Y. Directed Evolution of Group I Intron, SMBE 2023, (Ferrara, Italy), July 23 (2023). (Poster)
  6. Noma, T., Yokobayashi, Y. Towards Context-dependent Transgene Expression: Brain Cell-type Specific Gene Switches and Beyond, OIST-RIKEN Brain Symposium 2023, (Okinawa, Japan), Aug 22 (2023). (Poster)
  7. Yokobayashi, Y. Cell-Free Riboswitches: Engineering and Applications, On-chip Biotechnologies Seminar, (Online), Oct 16 (2023). (Oral)
  8. Hauf, S., Yokobayashi, Y. Control of Nucleic Acid Phase Separation using Aptamers, ISNAC 2023, (Miyazaki, Japan), Nov 1 (2023). (Oral)
  9. Noma, T., Yokobayashi, Y. Directed Evolution of Group I Intron in Mammalian Cells, ISNAC 2023, (Miyazaki, Japan), Nov 1 (2023). (Poster)
  10. Zhu, B., Yokobayashi, Y. Programmable Macroscopic Self-Assembly of LNA-Decorated Shape-Controlled Hydrogel Blocks, ISNAC 2023, (Miyazaki, Japan), Nov 1 (2023). (Poster)
  11. Nomura, Y., Yokobayashi, Y. A Ligase Ribozymes with a Small Catalytic Core, ISNAC 2023, (Miyazaki, Japan), Nov 1 (2023). (Poster)
  12. Noma, T., Yokobayashi, Y. Directed Evolution of Self-Splicing Ribozyme in Mammalian Cells, Library Design for Protein Engineering 2023, (Okinawa, Japan), Nov 7 (2023). (Poster)
  13. Yokobayashi, Y. Engineering functional RNAs in cells and test tubes, Library Design for Protein Engineering 2023, (Okinawa, Japan), Nov 8 (2023). (Oral, invited)
  14. Yokobayashi, Y. High-throughput analysis and exploration of ribozyme fitness landscapes, OIST x JST Joint Workshop: Biomolecular Super Assembly, (Okinawa, Japan), Jan 19 (2024). (Oral)
  15. 福永 圭佑, Dhamodharan Venugopal, 宮平 奈央, 野村 陽子, Mustafina Kamila, 大住 康晃, 高山 耕輔, 金井 章, 横林 洋平, 「低分子化合物に結合するRNAアプタマーを用いた動物細胞機能の制御」, 日本化学会 第104春季年会(2024), 日本大学理工学部 船橋キャンパス, Mar 18-21 (2024). (Poster)
  16. 福永 圭佑, 松浦 友亮, 横林 洋平, Engineering RNA-based switches for gene expression control. 核酸化学若手フォーラム2023, 宮崎市民プラザ, Oct 31 (2023). (Oral)
  17. 福永 圭佑, 勝木 陸, 横林 洋平, 松浦 友亮, 「セルフリーシステムを用いた合成リボスイッチのプロトタイピング」, 「細胞を創る」研究会 16.0, 東京大学駒場キャンパス, Sep 25-26 (2023). (Poster)
  18. 中島 ももか, 寺本 岳大, 横林 洋平, 福永 圭佑, 角田 佳充, 「高いRNA結合特異性を持つ2種類のL7Ae変異体-RNA複合体の結晶構造解析」, 学会創立100周年記念 日本農芸化学会 中四国・西日本支部 合同大会（中四国支部第66回講演会、西日本支部第347回講演会）, 高知県立県民文化ホール, Sep 21-22 (2023). (Poster).
  19. 福永 圭佑, Dhamodharan Venugopal, 宮平 奈央, 野村 陽子, Mustafina Kamila, 大住 康晃, 高山 耕輔, 金井 章, 横林 洋平, 「低分子化合物に結合するアプタマーを用いた哺乳類細胞および動物内RNA機能の制御」, 第17回バイオ関連化学シンポジウム, 東京理科大学・野田キャンパス薬学部, Sep 8-10 (2023). (Oral)
  20. 福永 圭佑, Dhamodharan Venugopal, 宮平 奈央, 野村 陽子, Mustafina Kamila, 大住 康晃, 高山 耕輔, 金井 章, 横林 洋平, Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals. 日本核酸医薬学会第8回年会, 名古屋大学・東山キャンパス・豊田講堂, Jul 11-14 (2023). (Poster)
  21. Keisuke Fukunaga, Dhamodharan Venugopal, Nao Miyahira, Yoko Nomura, Kamila Mustafina, Yasuaki Oosumi, Kosuke Takayama, Akira Kanai, and Yohei Yokobayashi, Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals. 第24回⽇本RNA学会年会, 那覇文化芸術劇場なはーと, Jul 5 (2023). (Poster)
  22. 中島 ももか, 寺本 岳大, 横林 洋平, 福永 圭佑, 角田 佳充, 「高いRNA結合特異性を持つL7Ae変異体-RNA複合体のX線結晶構造解析」, 第60回化学関連支部合同九州大会, 北九州国際会議場, Jul 1 (2023). (Poster)
  23. Keisuke Fukunaga, Dhamodharan Venugopal, Nao Miyahira, Yoko Nomura, Kamila Mustafina, Yasuaki Oosumi, Kosuke Takayama, Akira Kanai, Yohei Yokobayashi. Small Molecule Aptamer for Regulating RNA functions in Mammalian Cells and Animals. RNA2023, Suntec Convention & Exhibition Centre (Singapore), May 30-Jun 4 (2023). (Poster)

5. Intellectual Property Rights and Other Specific Achievements

A patent application was filed on the novel aptamer applications described above.

6. Meetings and Events

6.1 Nucleic Acids Modifications and Polymer Hybrids for Nanobiotechnology

  • Date: Oct 25, 2023
  • Venue: OIST Campus Lab3
  • Speaker: Prof. Subha R. Das (Carnegie Mellon University)

6.2 OIST x JST Joint Workshop: Biomolecular Super Assembly

  • Date: Jan 19, 2024
  • Venue: OIST Campus Lab1 (B250)
  • Co-organizers: Prof. Akihiro Kusumi (OIST), Prof. Hiroyuki Noji (The University of Tokyo, JST)
  • Speakers:

7. Other

Degree awarded

  • Rachapun Rotrattanadumrong

Rotation students

  • Muhammad Hamzah
  • Anna Klarkowska

Research interns and visiting students

  • Rosa Marquez Costa (University of Valencia) Apr 2023 - Sep 2023
  • Pouryia Bayat (University of Toronto) Jun 2023 - Sep 2023

Outreach activities

  • Yoko Nomura, Panelist at esse-sense Forum 2023, Sep 22, 2023, (Tokyo, Japan)
  • Yoko Nomura, Speaker in Mingei Seminar at The Japan Folk Crafts Museium, Oct 15, 2023, (Osaka Japan) 

External funding and support

  • Yohei Yokobayashi: Undisclosed industry partner (continuing, PI), JSPS Summer Internship host, AMED/BINDS research support.
  • Narae Kim: KAKENHI Young Resercher (continuing, PI) 22K15000
  • Samuel Hauf: Walter Benjamin Fellowship (continuing, through Nov 2023)
  • Rachapun (Gear) Rotrattanadumrong: JSPS Predoctoral Fellowship (continuing, through Aug 2023)